runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: Evidence for promoter context-dependent activity of Cbfa proteins

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2 alphaA) transc ription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts, This study addresses the func tional involvement of Cbfa sites in expression of the Gallus BSP gene, Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa D NA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells , Each of the Cbfa factors mediated repression of the wild-type BSP promote r, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not obser ved in BSP promoters in which Cbfa sites were deleted or mutated, Expressio n of the endogenous BSP gene in Gallus osteoblasts was similarly downregula ted by forced expression of Cbfa factors. Our data indicate that Cbfa repre ssion of the BSP promoter does not involve the transducin-like enhancer (TL E) proteins, Neither coexpression of TLE1 or TLE2 nor the absence of the TL E interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity, However, removal of the C terminus of Cbfa1 (amino acids 362 to 5 13) relieved suppression of the BSP promoter, Our results, together with th e evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.